Systems and methods for rapid nucleic acid extraction, purification and analysis from bone and tooth

ABSTRACT

Disclosed are processes and kits for rapid nucleic acid extraction from a nucleic acid-containing material, such as a bone, tooth or semen sample. For bone and tooth process involves providing the nucleic acid-containing material in a form suitable for nucleic acid extraction, adding a lysis buffer to the nucleic acid-containing material to obtain a mixture, mixing the mixture in a manner equivalent for about 30 seconds or longer and separating the mixture by centrifugation to obtain a liquid supernatant. The liquid supernatant contains the extracted nucleic acids which can be used for analysis including STR profiling by conventional or rapid DNA analysis. For semen the processes and kits involve applying an appropriate amount of sperm disruptive agent.

FIELD OF INVENTION

The invention relates to systems and processes for nucleic acidextraction, purification, and analysis. More specifically, in oneaspect, the invention relates to rapid purification and analysis ofnucleic acids from samples including bone, tooth, and semen samplesobtained from forensic, missing persons, and mass casualty settings.

BACKGROUND

In forensic science, a major objective is to identify sources ofbiological evidence such as bone, tooth, blood, semen, saliva, and hairsfound at the scene of crimes, terrorist activities and attacks, and massdisaster sites. DNA profiling has provided a means of identifying thesource of such material with a very high degree of certainty. In theforensic science community today, most DNA profiling methods are basedon the amplification of small regions of the human genome containing aclass of repeated DNA stretches known as Short Tandem Repeats (STRs).The unit length of a given STR typically ranges between 2-10 base pairs,and STRs generally fall within non-coding and flanking sequences butoccasionally within coding regions. There are several hundred thousandSTR loci in the human genome, occurring on average every 6-10 kb andappearing to be highly polymorphic. The number of repeats at a given STRsite or locus in the genome is characteristic of the cells of anindividual, and by determining the number of repeats at several STRloci, a characteristic “DNA fingerprint” (also referred to as an “STRprofile” or “DNA ID”) of an individual can be generated.

STR profiles are generated by a series of three basic processes. First,DNA is purified from the cells on a sample collection device orsubstrate, (e.g., a swab). This involves breaking open the cells to freethe DNA and then removing proteins, other biomolecules, and cellulardebris to generate a purified or partially purified DNA solution.Second, the set of STR loci are copied (amplified) using a process knownas Polymerase Chain Reaction (PCR). Primers that bind to target STR generegions are labeled with fluorescent dyes so amplified fragmentscontaining said primers can be detected by laser-induced fluorescence. Alarge number of dyes (greater than 50) are available for use influorescence excitation applications. These dyes include those from thefluorescein, rhodamine AlexaFluor, Biodipy, Coumarin, and Cyanine dyefamilies. Furthermore, quenchers are also available for labelingoligonucleotide sequences to minimize background fluorescence. Dyes withemission maxima from 410 nm (Cascade Blue) to 775 nm (Alexa Fluor 750)are available and can be used. Dyes ranging between 500 nm to 700 nmhave the advantage of being in the visible spectrum and can be detectedusing conventional photomultiplier tubes. The broad range of availabledyes allows selection of dye sets that have emission wavelengths thatare spread across the detection range. Detection systems capable ofdistinguishing many dyes have been reported for flow cytometryapplications (see, Perfetto et al., Nat. Rev. Immunol. 2004, 4, 648-55;and Robinson et al., Proc of SPIE 2005, 5692, 359-365).

Fluorescent dyes have peak excitation wavelengths that are typically 20to 50 nm blue-shifted from their peak emission wavelength. As a result,use of dyes over a wide range of emission wavelengths may require theuse of multiple excitation sources, with excitation wavelengths toachieve efficient excitation of the dyes over the emission wavelengthrange. Alternatively, energy transfer dyes can be utilized to enable asingle laser, with a single emission wavelength, to be used for excitingall dyes of interest. This is achieved by attaching an energy transfermoiety to the dye label. This moiety is typically another fluorescentdye with an absorption wavelength that is compatible with the excitationwavelength of the light source (e.g. laser). Placement of this absorberin close proximity with an emitter allows the absorbed energy to betransferred from the absorber to the emitter, allowing for moreefficient excitation of the long wavelength dyes (Ju et al., Proc NatlAcad Sci USA 1995, 92, 4347-51). A fourth dye is used as a standardmarker for determining the size of the STR fragments.

Third, the size of the copied STR fragments is determined byelectrophoresis. The STR fragments are pulled by an electric currentthrough a gel-like substance, and the smaller fragments travel morequickly through the gel than the larger fragments. At a detectionwindow, a laser is used to excite the four fluorescent dyes. The dyesemit light, which is then detected and used to determine the size ofeach STR fragment. An Expert System, operated either automatically ormanually, analyses the size pattern and generates the final STRprofile—the individual's DNA fingerprint. Note that the three basicsteps of DNA fingerprint generation may be modified; for example, cellsin a sample may be lysed, and the resulting cell extract moved toamplification (without the DNA purification). Similarly, amplified DNAfragments can be identified by methods including mass spectroscopy.

STR analysis was first applied in the early 1990's and has since becomea major tool in the forensic armamentarium with a growing set ofapplications including law enforcement, paternity testing, humanidentification in mass disasters, immigration and sexual assault cases,unclaimed minor (child) verification and human trafficking. In theUnited States, STR profiles generated from selected human samples arecollected in the Combined DNA Index System (CODIS). This electronicdatabase was established in 1997 by the FBI and standardized to a set of13 tetrameric STR loci for data submission, and, in January 2017, thenumber of core loci to be utilized in FBI databases expanded to 20.Other STR databases outside the US contain overlapping subsets of theCODIS loci. In order to generate a DNA profile, a critical first step isto obtain DNA from the sample of interest.

Forensic samples present special challenges. The inventions presentedhere provide solutions for bone, tooth, and semen samples, among themost difficult for processing in forensic laboratories. For bone andtooth, there is no universal protocol that allows for extraction of DNAfrom materials at different stages of degradation. The efficiency of DNAextraction, the level of demineralization, the problem of low content ofDNA in the sample, the degree of contamination by inhibitors all presentsignificant challenges. At least two types of semen samples arerelevant. First, a semen stain containing a relatively pure populationof sperm may be found on, for example, an article of clothing or on theexposed skin of a rape victim. Second, Sexual Assault Kit samples (SAKs)often contain vaginal swabs or cervical swabs—such swabs may containmixtures of vaginal and cervical cells (including vaginal and cervicalepithelial cells) and sperm. In addition, both semen sample types maycontain cells from more than one male—whether derived from a consensualpartner or an additional rapist. Separation of male and females cellsand deconvolution of mixtures also present challenges.

A related challenge relates to the development and use of processingprotocols, including processing protocols for Rapid DNA Identification.Processing protocols are very important in many different fields.Processing protocols are created and followed to ensure that similarsample types are processed in the same way. The use of such protocolshelps provide consistent processing results, and where the results arenot consistent, ensures that the differences are not attributable tovariances caused by the processing itself. With regard to STR analysis,certain protocols must be followed not only as a matter of soundscientific principles, but also to stay within compliance with federalregulations. Protocols that have been developed for manual processing orwithout regard to field-forward use or rapid processing may be entirelyunsuitable to forensic use, including field forward forensics. Wepresent herein novel inventive protocols that solve the unique problemspresented by the necessity of automated processes, fast protocols, andforensic samples.

There are still major challenges in efficient nucleic acid purificationfrom certain biological evidence such as bone. Bone is a good source ofnucleic acid under scenarios where the biological evidence has beenexposed to a variety of environmental conditions because the bone matrixprotects nucleic acids from degradation. Current methods of nucleic acidextraction from bone are generally cumbersome, lengthy, and inefficientin that it takes hours or even days to obtain quality nucleic acidssuitable for analysis. Bone and tooth purification protocols are basedon extraction and purification of DNA from bone or tooth powderpulverized by a freezer mill or blending cup (Loreille et al, FSIG 1(2007), 191-195; Johnston and Stephenson, JFS 61 (2016), 898-902;Turingan et al, Inv. Genetics 7 (2016), bone slices generated using arotary dental saw or its equivalent (Kitayama et al, Legal Med 12(2010), 84-89), and related protocols. The need to initiate the processwith powder requires cumbersome equipment and limits the techniques tosophisticated laboratories. In addition, pulverization equipment anddental cutting discs can cause the production of airborne biologicalmaterial, with attendant health risks to operators as well as attendantrisks of sample-to-sample contamination. Furthermore, extensivedemineralization of the bone powder or bone slices, often requiringovernight demineralization at elevated temperatures with agitation (See,e.g., Lee H Y et al., Forensic Science International: Genetics, 4 (2010)275-280) also take time and sophisticated equipment. Finally, theprotocols of the prior art are based on large volumes ofdemineralization buffer relative to the amount of the bone powder to bedemineralized and rely on complex reagent mixes. The large volumes ofdemineralization buffer requires concentration (often using aconcentration membrane) in order to obtain an appropriate volume forsubsequent processing (e.g. purification) and analysis. Concentration isyet another step that adds time and complexity to the overall process.Taken together, it is no wonder why the time to generation of STRprofiles from bone is a major problem in law enforcement forensiclaboratories, coroners' offices, and Offices of Chief Medical Examiners.

To date, STR analysis of bone and tooth powder has been performed inconventional laboratories. The investment in labor, instrumentation, andtime has dramatically limited the utility of this approach. Manyforensic laboratories send out bone and tooth processing to highlyspecialized laboratories that can take a year or more to return DNAfingerprint results and have limited capacity to do even that. As aresult, it is estimated that 40-80,000 unidentified corpses are now inmedical examiners' and coroners' offices, and many are buried orcremated before they can be identified. Similarly, even a relativelysmall disaster such as a plane crash can take years for body parts to beidentified by forensic laboratories. When large mass disasters occur,bodies may be unidentified for years or decades. The 2004 Indian Oceanearthquake and tsunami is a tragic example—more than a decade later,thousands of victims remain unidentified.

Both conventional and rapid DNA analysis systems would both benefitgreatly from faster, simpler, and more efficient processing of samplesof interest. The processes and kits in the present invention aresurprising in the following aspects. First, in the present invention,the need for generating bone or tooth powder has been eliminated.Unexpectedly, a few blows of a hammer or a mortar and pestle or a brick(or similar material) macerate the sample sufficiently for furtherprocessing. The surface area generated for demineralization by hammeringsurprisingly generates sufficient surface area to enable efficientdemineralization while avoiding the generation of airborne boneparticles. Second, the process of demineralization of bone samples takesless than 60 minutes, with ten minutes being generally applicable andonly one minute being sufficient in many instances. For tooth, theprocess of demineralization takes approximately 180 minutes, with tenminutes being sufficient in many instances. Third, prior art protocolsrequire overnight demineralization to extract nucleic acids from bonepowder and slices, often with heating steps that can damage DNA. SeeHiggins et al, Forensic Sci Med Pathol 2014, 10:56-61; Amory et al.,Forensic Science International: Genetics 6 (2012) 398-406; Loreille etal. Forensic Science International: Genetics 1 (2007) 191-195; and Leeet al. Forensic Science International: Genetics 4 (2010) 275-280. In theLoreille method, total dissolution of bone powder using a large volumeof buffer was necessary for analysis, and organic extraction forpurifying DNA followed by sample concentration was necessary. TheLoreille method, therefore, requires excessive sample manipulation andtime, both of which limit the process to a sophisticated andwell-equipped laboratory and dramatically limit the processing capacityof the laboratory at that. Higgins is a modification of the Loreillemethod which, while using lower volume assays, still requires anovernight incubation and laborious purification. In addition, theprotocol requires the use of carrier RNA which is an additional reagentand involves additional processing steps. The carrier RNA is taught tobe added to increase DNA yield, however the addition does not result inenhanced DNA recovery. The Lee method uses more reagents and is evenmore complex than either Loreille or Higgins. Fourth, standard methodsuse large volumes of demineralization buffer, which makes handling ofthe solutions and subsequent DNA concentration and purification morechallenging for conventional and Rapid DNA analysis. Taken together,none of these methods are appropriate for field-forward Rapid DNAIdentification.

Fourth, the processes of the invention also use specialized reagents,including demineralization reagents. In the present invention, thedemineralization solution contains only minimal EDTA buffer volume andinvolves a quick demineralization prior to downstream purification frombone and STR profile generation. We demonstrate herein unexpectedresults, including that demineralization solutions that may containlysis buffer/detergent and proteinase K need not contain EDTA in a highinitial volume (˜15 ml for complete dissolution of 1 g sample). Priorart methods using lysis buffer/detergent and high initial volumes ofEDTA teach the use of a long incubation step followed by sampleconcentration prior to DNA purification. We demonstrate the unexpectedfinding that these steps are not necessary. Similarly, prior artprotocols for DNA extraction from demineralized bone and tooth involvean organic extraction method. Prior art protocols for isolating nucleicacid from semen samples require the use of complex reagents and longprocessing steps necessary to isolate and lyse the cells from the mucousenvironment in which they usually are found, including centrifugation,washing, and incubation steps. We demonstrate herein novel, unexpectedprotocols for isolating nucleic acid from bone, teeth and semen samplesthat shatter the conventional wisdom.

The simplified solutions and protocols of this invention not only reducereagent and material cost but substantially decrease processing time andpotential for sample loss during extensive manipulation. Indeed, thisinvention is based, in part, on the unexpected finding that there is noneed to completely dissolve the sample for a total demineralization withlonger incubation and high buffer volume for generation of callable STRprofiles. The solutions and protocols of this invention are easilyadapted for use in integrated biochip and instrument systems which arecapable of sample-in to results-out processing.

The solutions and protocols of this invention are able to extractnucleic acids in sufficient quantities to generate STR profiles that canbe uploaded into the CODIS database. For example, from 15 fresh bonesamples, using the protocols of the invention, we obtained a nucleicacid yield of 4.2 ng-108.1 ng per mg of bone. In addition, from 12 freshtooth samples, we obtained a nucleic acid yield of 0.1 ng-2.7 ng per mgof tooth root. Finally, the solutions and protocols of this inventionmay be adapted for use in high and low DNA content integrated Rapid DNAIdentification systems (using A-Chips and I-Chips, respectively),including those capable of sample-in to results-out processing.Automated data processing and automated Expert System analysis may alsobe incorporated into the Rapid DNA Identification system.

With regard to protocols for DNA extraction from semen, faster, simpler,more efficient methods have been developed that can be utilized for bothconventional and Rapid DNA analysis systems. Standard DNA purificationmethods for somatic cells are ineffective for lysing sperm cells due totheir high degree of nuclear compaction and presence of protectivemembranes rich in disulfide bonds. Modifications to standard lysismethods include addition of a strong reducing agents to disrupt thesedisulfide bonds that impede lysis. These alternative methods primarilyrely on lengthy chaotropic digestion with proteinase K at elevatedtemperatures and incubation with reducing agent such as dithiothreitol(DTT) to allow consequent isolation and purification of DNA. Rapidmethods of mammalian sperm DNA isolation have also been conducted usingsteel beads for homogenization at room temperature for 5 minutes in thepresence of chaotropic lysis buffer and addition oftris(2-carboxyethyl)phosphine (TCEP) instead of DTT (Wu et. al.Biotechniques. 2014; 58(6): 293-300). Although this method substantiallydecreased time and complexity by eliminating heat and slow incubationand uses an odorless sperm disruption agent, mechanical beadhomogenization techniques are not always available and still requirehighly skilled technicians, sophisticated equipment, and clean-up/beadisolation for downstream processing. In short—a laboratory environment.In the present invention, semen samples are collected on a swab (eitherprepared from neat semen, dried semen on fabric, carpet, skin, tile orother substrates, or isolated spermatozoa from sexual assault kits) andthen a sperm disruption agent (e.g. 150 mM DTT or 50 mM TCEP) is simplyadded. Incubation of semen on swab at room temperature for 10 minutes,and in many instances for as little as 1 minute, was enough to generateDNA fingerprints using the ANDE Rapid DNA system.

In sexual assault kits, vaginal swabs often contain cells from more thanone contributor. Often the swabs will contain cells from the femalevictim and the male perpetrator. Accordingly, to avoid “mixed” DNA IDs(in which the DNA IDs from two or more individuals in present),spermatozoa may be separated from the vaginal epithelial cells;processing to achieve this separation is necessary prior to DNAextraction and/or purification steps. A differential lysis method wasdescribed in 1985 by Gill and colleagues which relies on separatingintact sperm from the DNA of lysed epithelial cells. Severalmodifications to the method to reduce time and improve efficiency havebeen made over the years, but the fundamental concept and process hasremained unchanged. Differential extraction is time-consuming,laborious, and required a sophisticated laboratory and highly-skilledtechnicians. Physical separation of the intact sperm cells from thelysed female fraction is achieved by centrifugation and repeated washingof the pelleted sperm cells. Alternative methods eliminate thetime-consuming wash steps by selective degradation of soluble DNA usingnuclease enzyme (Garvin et al Investigative Genetics 2012 3:25). Acommercially available kit, Sperm Erase (Paternity Testing Corporation)utilizes the latter approach. Processing of SAKs using prior art methodsrequire between 2 to 5 hours from the initial epithelial cell lysis tothe generation of sperm fraction.

The present invention provides a rapid method for lysing epithelialcells which unexpectedly can be performed in ten seconds at roomtemperature; previous approaches require at least one hour and typicallyrequire heat. Furthermore, the present invention provides a methodwherein both physical separation and chemical degradation techniqueshave been efficiently combined to allow effective isolation andpurification of sperm cell DNA while reducing overall process time.

In one method of the present invention, the sperm pellet is washed oncewith wash buffer that is optimized for nuclease activity Eliminating therepeated wash steps not only improves time but also reduces potentialsperm cell loss. The wash buffer is stable at room temperature andprimes the reaction for the addition of nuclease, hence, one lessreagent in an SAK processing kit. The nuclease degrades any soluble DNAand a stop solution containing 0.5M EDTA is then added to deactivate theenzyme. To allow lysis of the sperm cells, a sperm disruption agent(with final concentrations of approximately 150 mM DTT or 50 mM TCEP) isadded to the mixture prior to collecting the liquid with a swab forRapid DNA analysis. Generation of male fraction was completed in 32minutes. Decreasing the centrifugation time for initial separation andalso decreasing the time to quench the activity of the nuclease allowedgeneration of sperm fraction for analysis in approximately 22 minutes.

Furthermore, the process steps have been simplified so that they may beperformed by non-technical personnel. In fact, the work can be performedoutside the forensic lab, by Sexual Assault Nurse Examiners, Forensicnurses, or other hospital personnel. If the work is performed at thehospital (even in a hospital lab), the transport time to the policestation/forensic lab is eliminated. Taken together, the teachings of thepresent invention all rapid generation of an actionable DNA ID result.By generating the result quickly, law enforcement investigations canproceed more quickly leading to suspect apprehension and exoneration ofthe innocent. As many criminals are recidivists (e.g. the typical rapistcommits more than 10 rapes before being apprehended), the teachingsherein can be expected to dramatically reduce crime and improve publicsafety.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment, we provide a process for extracting nucleic acidsfrom a bone or tooth sample, comprising providing the sample in a formsuitable for nucleic acid extraction; adding a demineralization bufferto the nucleic acid-containing material to obtain a mixture; mixing themixture vigorously; and separating the mixture to obtain a liquidsupernatant; wherein the liquid supernatant contains the extractednucleic acids. In related embodiments, the separation step does notinvolve concentrating the nucleic acid and the demineralization does notresult in complete dissolution of the bone sample.

In another embodiment we provide a process for extracting nucleic acidsfrom a bone or tooth sample, comprising: providing the sample in a formsuitable for nucleic acid extraction; adding a less than 500 μl 0.5MEDTA or equivalent demineralization buffer to the nucleicacid-containing material to obtain a mixture; mixing the mixturevigorously; incubating the mixture for at least one minute withoutadding heat; separating the mixture to obtain a liquid supernatantwithout concentration wherein the liquid supernatant contains theextracted nucleic acids.

In yet another embodiment, we provide a process for determining STRprofiles of nucleic acids from a nucleic acid-containing material,comprising: providing the nucleic acid-containing material in a formsuitable for nucleic acid extraction; adding a demineralization bufferto the nucleic acid-containing material to obtain a mixture; mixing themixture vigorously; separating the mixture to obtain a liquidsupernatant; wherein the liquid supernatant contains the extractednucleic acids; and subjecting a portion of the liquid supernatant to anucleic acid analysis to determine the STR profile of the nucleic acidsfrom the nucleic acid-containing material.

In another embodiment we provide a process and kits for determining STRprofiles of nucleic acids from a semen sample, comprising: collectingsemen samples onto a transfer medium; applying an appropriate amount ofa sperm disruptive agent onto the transfer medium. In relatedembodiments the transfer medium may be inserting the swab into a rapidDNA analysis system or otherwise processed for STR analysis.

In another embodiment, A process and kits for isolating femaleepithelial cells from sperm cells in post-coital vaginal swabscomprising: lysis of epithelial cells from transfer medium; physicalseparation of intact sperm cells from lysed epithelial cells; removal ofthe aqueous female fraction from the intact sperm cells; washing of thepelleted intact sperm cells by wash buffer; degradation of any solubleDNA by nuclease treatment; inactivation of nuclease, lysis of spermcells by a sperm disruption agent (e.g. DTT or TCEP, other chemicals, orphysical methods); and collection of the lysed sperm fraction.

In yet another embodiment, we provide a process for extracting nucleicacids from a sample suspected of containing sperm, the processcomprising: inserting said sample into a sample container containing afirst lysis solution and agitating to make a first mixture; centrifugingsaid first mixture; washing the pellet in said first container withbuffer; second centrifuging the first container to repelletize any spermpresent in said sample; transfering at least a portion of the sperm to asecond container containing nuclease in a sufficient amount, and undersufficient conditions to degrade DNA; adding a sperm disruption agent;recovering the disrupted sperm fraction for further processing.

As will be apparent to persons of ordinary skill in the art theinventive processes for extraction, purification and analysis of nucleicacids result in outputs that are suitable for further analysis,including without limitation, downstream processing in integratedbiochips. The embodiments mentioned in this summary are not intended tolimit the claims of this or of any related or unrelated application.Other aspects, embodiments, modifications to and features of the claimedinvention will be apparent to persons of ordinary skill in the art inview of the disclosures herein.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a full 16-locus STR profile of nucleic acid purified from200 mg freeze-milled bone powder of fresh femur sample. Sample wasdemineralized for 1 minute and analyzed in an A-Chip.

FIG. 2 shows a full 16-locus STR profile of nucleic acid purified from10 mg freeze-milled bone powder of fresh femur sample. Sample wasdemineralized for 1 minute and analyzed in an I-Chip.

FIG. 3 shows a full 16-locus STR profile of nucleic acid purified from500 mg freeze-milled bone powder of an artificially aged femur sample.Bone was soaked for 4 days in saltwater at 84° F., cleaned and dried,demineralized for 1 minute, and analyzed in an I-Chip.

FIG. 4 shows a full 16-locus STR profile of nucleic acid purified from200 mg freeze-milled bone powder of an artificially aged femur sample.Bone was soaked for 4 days in saltwater at 84° F., cleaned and dried,demineralized for 1 minute, and analyzed in an I-Chip.

FIG. 5 shows a full 16-locus STR profile of nucleic acid purified from500 mg hammered bone fragments of an artificially aged decomposed femursample. Bone was soaked for 4 days in saltwater at 84° F., cleaned anddried, demineralized for 1 minute, and analyzed in an I-Chip.

FIG. 6 shows a full 16-locus STR profile of nucleic acid purified from200 mg hammered bone fragments of an artificially aged decomposed femursample. Bone was soaked for 4 days in saltwater at 84° F., cleaned anddried, demineralized for 1 minute, and analyzed in an I-Chip.

FIG. 7 shows a full 16-locus STR profile of nucleic acid purified from500 mg freeze-milled bone powder of an artificially aged femur sampleusing the nucleic acid extraction method as reported by Loreille et al.Forensic Science International: Genetics 1 (2007) 191-195. Bone wassoaked for 4 days in saltwater at 84° F., cleaned and dried, andanalyzed in an I-Chip.

FIG. 8 shows a full 16-locus STR profile of nucleic acid purified from200 mg freeze-milled root of fresh molar tooth sample. Sample wasdemineralized for 10 minutes and analyzed in an I-Chip.

FIG. 9 shows a full 16-locus STR profile of nucleic acid purified fromdried semen stain on denim and analyzed in an I-Chip.

FIG. 10 shows a full 16-locus STR profile of nucleic acid purified fromdried semen on cotton and analyzed in an I-Chip.

FIG. 11 shows a full 27-locus STR profile of nucleic acid purified from5 mg freeze-milled bone powder of fresh femur sample. Sample wasdemineralized for 1 minute and analyzed in an A-Chip.

FIG. 12 shows a full 27-locus STR profile of nucleic acid purified from5 mg hammered bone fragments of fresh femur sample. Sample wasdemineralized for 1 minute and analyzed in an A-Chip.

FIG. 13 shows a full 27-locus STR profile of nucleic acid purified from5 mg freeze-milled bone powder of fresh femur sample and using only 15μl of 120 μl bone demineralized solution for analysis in an I-Chip. Thesample input is theoretically equivalent to 0.6 mg sample. Sample wasdemineralized for 1 minute.

FIG. 14 shows a full 27-locus STR profile of nucleic acid purified from5 mg hammered bone fragments of fresh femur sample and using only 15 μlof 120 μl bone demineralized solution for analysis in an I-Chip. Thesample input is theoretically equivalent to 0.6 mg sample. Sample wasdemineralized for 1 minute.

FIG. 15 shows a full 27-locus STR profile of nucleic acid purified from5 mg hammered fresh distal phalanx bone sample and using only 15 μl of120 μl bone demineralized solution for analysis in an I-Chip. The sampleinput is theoretically equivalent to 0.6 mg sample. Sample wasdemineralized for 1 minute.

FIG. 16 shows a full 16-locus STR profile of nucleic acid purified from200 mg freeze-milled fresh tooth root (not subjected to any form ofdegradation). Tooth was demineralized for 1 minute and analyzed in anI-Chip.

FIG. 17 shows a full 16-locus STR profile of nucleic acid purified from200 mg freeze-milled root of an artificially aged molar tooth sample.Tooth was soaked for 1 month in saltwater at 84° F., cleaned and driedprior to separating the root from crown, demineralized for 10 minutes,and analyzed in I-Chip.

FIG. 18 shows a full 16-locus STR profile of nucleic acid purified from200 mg milled root of a forensic molar tooth sample. Tooth was foundburied in soil for at least 2 years, cleaned and dried prior toseparating the root from crown, demineralized for 180 minutes, andanalyzed in an I-Chip.

FIG. 19 shows a full 27-locus STR profile of nucleic acid purified from10 mg hammered root fragments of fresh molar tooth sample. Sample wasdemineralized for 10 minutes and analyzed in an I-Chip.

FIG. 20 shows a full 27-locus STR profile of nucleic acid purified from10 mg hammered root fragments of fresh incisor tooth sample. Sample wasdemineralized for 10 minutes and analyzed in an I-Chip.

FIG. 21 shows a partial STR profile (24 of 27 loci) of nucleic acidpurified from 200 mg hammered root fragments of aged molar tooth sample.Tooth was extracted from human body placed 120 days above field ground,cleaned and dried prior to separating the root from crown, demineralizedfor 180 minutes, and analyzed in I-Chip.

FIG. 22 is a plot showing the average amount of purified DNA (innanograms) as a function of the volume of demineralization buffer (inmicroliters) added to a 500 mg bone powder sample. Data was taken fromquadruplicate measurements, with error bars representing 1STDEV. Thisdata led us to believe that we can use less buffer for less bone (ortooth) as starting material.

FIG. 23 is a time-course data showing demineralization of bone measuredas purified DNA (in nanograms) as a function of time. The datademonstrates that overnight demineralization is not required for STRanalysis. Demineralization times of one minute are sufficient for STRanalysis using the inventive compositions and methods of this invention.

FIG. 24 is a plot showing the average amount of purified DNA (innanograms) using EDTA with and without detergents. This data is acomparison of simplified demineralization buffers that can be used inthis invention. Data also shows that a simple 0.5M EDTA buffer issufficient to demineralize bone and obtain sufficient amounts of DNA forSTR analysis.

FIG. 25 shows the 32-minute protocol developed for processing sexualassault kits.

FIG. 26 shows the 22-minute protocol developed for processing sexualassault kits. Centrifugation time to separate intact sperm cells fromthe lysed epithelial fraction has been reduced from 5 minutes to 2minutes at 20000×g. Incubation time for the stop solution to deactivatethe nuclease was also reduced from 10 minutes to 3 minutes at 56 C.

FIG. 27 lists the components of the SAK kit based on developedprotocols.

FIG. 28 shows a full 27-locus male STR profile of nucleic acid purifiedfrom male fraction isolated from vaginal swab (Donor couple A/B)collected 24 hrs post-coitus using the 32-minute protocol. Male genotypeis indicated by blue arrows based on processed male buccal referenceswab. Female genotype is indicated by red arrows based on processedfemale buccal reference swab.

FIG. 29 shows a full 27-locus male STR profile of nucleic acid purifiedfrom male fraction isolated from vaginal swab (Donor couple A/B)collected 48 hrs post-coitus using the 32-minute protocol. Male genotypeis indicated by blue arrows based on processed male buccal referenceswab. Female genotype is indicated by red arrows based on processedfemale buccal reference swab.

FIG. 30 shows a full 27-locus male STR profile of nucleic acid purifiedfrom male fraction isolated from vaginal swab (Donor couple A/B)collected 72 hrs post-coitus using the 32-minute protocol. Male genotypeis indicated by blue arrows based on processed male buccal referenceswab. Female genotype is indicated by red arrows based on processedfemale buccal reference swab.

FIG. 31 shows a partial 27-locus male STR profile (24 of 27) of nucleicacid purified from male fraction isolated from vaginal swab (Donorcouple C/D) collected 72 hrs post-coitus using the 32-minute protocol.STR profile is missing alleles for D2S441, D10S1248, and TPDX. Malegenotype is indicated by blue arrows based on processed male buccalreference swab. Female genotype is indicated by red arrows based onprocessed female buccal reference swab.

FIG. 32 shows a partial 27-locus male and female mixture (with femaleminor) STR profile of nucleic acid purified from male fraction isolatedfrom vaginal swab (Donor couple E/F) collected 72 hrs post-coitus usingthe 32-minute protocol. Male STR profile is missing an allele for vWA.Male genotype is indicated by blue arrows based on processed male buccalreference swab. Female genotype is indicated by red arrows based onprocessed female buccal reference swab.

FIG. 33 shows a partial 27-locus male and female mixture (with femalemajor) STR profile of nucleic acid purified from male fraction isolatedfrom vaginal swab (Donor couple G/H) collected 72 hrs post-coitus usingthe 32-minute protocol. Male STR profile is missing alleles forD10S1248, PentaE, D5S818, CSF1PO, and SE33. Male genotype is indicatedby blue arrows based on processed male buccal reference swab. Femalegenotype is indicated by red arrows based on processed female buccalreference swab.

FIG. 34 shows a full 27-locus 1:1 male and female mixture STR profile ofnucleic acid purified from male fraction isolated from vaginal swab(Donor couple I/J) collected 72 hrs post-coitus using the 32-minuteprotocol. Male genotype is indicated by blue arrows based on processedmale buccal reference swab. Female genotype is indicated by red arrowsbased on processed female buccal reference swab.

FIG. 35 shows a full 27-locus male STR profile of nucleic acid purifiedfrom male fraction isolated from vaginal swab (Donor couple C/D)collected 72 hrs post-coitus using the 22-minute protocol. Male genotypeis indicated by blue arrows based on processed male buccal referenceswab. Female genotype is indicated by red arrows based on processedfemale buccal reference swab.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered simpler and faster processes forextracting and purifying nucleic acids from a nucleic acid-containingmaterial, particularly from bone, tooth, and semen (including vaginalswabs from Sexual Assault Kits). For bone and tooth, simple fragmentsgenerated by a hammer are suitable for processing, and bone/tooth powderis unnecessary. In one aspect, the invention is directed to a simplifiedand fast process for extracting nucleic acids from a nucleicacid-containing material. In another aspect, the invention is kits forperforming the simplified and rapid processes for extracting nucleicacids from a nucleic acid-containing material. In yet another aspect,the invention is an integrated process for rapid nucleic acid analysis,e.g., Rapid DNA Identification based on STR profiling, where the sampleprocessing, DNA extraction, purification, amplification, separation,detection, and analysis occur in a fast, efficient, and fully-integratedmanner.

The inventions herein described are further explained and enabled, andmay be used alone or in combination with other nucleic acid sequencingand sizing instruments, biochips and methods, co-owned with thisapplication as set forth in the following patents, each of which isincorporated by reference in its entirety herein: U.S. Pat. Nos.9,889,449 and 9,366,631 entitled “Integrated systems for the multiplexedamplification and detection of six and greater dye labeled fragments;U.S. Pat. Nos. 9,797,841 and 9,310,304 entitled “Methods andCompositions for Rapid multiplex amplification of STR loci,” U.S. Pat.Nos. 9,606,083; 9,523,656; 8,206,974; 8,173,417 entitled “RuggedizedApparatus for Analysis of Nucleic Acid and Proteins”; 9,550,985 entitled“Methods for Forensic DNA Quantitation”; U.S. Pat. Nos. 9,494,519 and8,425,861 entitled “Methods for Rapid Multiplexed Amplification ofTarget Nucleic Acids”; U.S. Pat. No. 8,018,593 entitled “IntegratedNucleic Acid Analysis”; U.S. Pat. Nos. 9,354,199; 9,314,795; 8,720,036entitled “Unitary Biochip Providing Sample-In to Results-Out Processingand Methods of Manufacture”; U.S. Pat. Nos. 9,174,210 and 9,012,208entitled “Nucleic Acid Purification”; U.S. Pat. Nos. 8,961,765 and8,858,770 entitled “Plastic Microfluidic Separation and DetectionPlatforms”.

Processes for Nucleic Acid Extraction from Bone and Tooth

In one aspect, the invention is a simplified and fast process forextracting nucleic acids from a bone or tooth, and the process comprisesthe steps of providing the nucleic-acid containing material sample in aform suitable for nucleic acid extraction, mixing vigorously the samplein a demineralization buffer, e.g., 0.5M EDTA, and separating themixture to obtain a liquid portion of the mixture which contains theextracted nucleic acids.

In some embodiments, bone and tooth samples may be fresh samples fromdonors with post-mortem interval (PMI) less than 8 hours or relativelyfresh samples from donors with post-mortem interval (PMI) of 2-6 days.In other embodiments, bone and tooth may be from casework, disaster,missing persons or related samples. Such samples may be obtained asdegraded as is the case, for example, decayed bodies in fresh water,salt water, or soil or after a fire or explosion. Bone and tooth samplesmay be years, decades, or centuries old, often exposed to the elements.In mass disasters, missing persons cases, terrorist and criminalincidents, and atrocities committed by dictatorial regimes, this abilityto generate STR profiles from such aged bone is critical.

In some embodiments, the samples are first cleaned and then grinded sothat the samples are in a form suitable for nucleic acid extraction. Forexample, the samples may be washed with water and scrubbed to removedebris, soaked in 10% bleach solution, and washed again with sterilewater before a final wash with 70% ethanol. After the final wash withethanol, the samples may be dried at room temperature and grinded, forexample, by freeze-mill to make powderized samples or hammering to makefragmented or granulated samples. Although not absolutely required,cleaning is advantageous in that it may remove large quantities ofmicrobial contaminants and, just as importantly, human DNA introduced byhuman handling or mixtures from two or more sample donors (if cleaningis not utilized, potential mixed profiles may be deconvolved manually orusing software). As will be discussed below, milling is not necessary,an important consideration when the work is to be performed outside thelaboratory.

In some embodiments, the bone or tooth sample may be milled andpowderized. By way of example, a piece of bone may be milled using manytechniques, for example using the SPEX Sample Prep 6870 LargeFreezer/Mill® (SPEX Sample Prep LLC, Metuchen, N.J.) following a 3-cycleprogram, 10 minute pre-cool, 2 minute run time, 2 minute cool time, andwith a rate of 10 cycles per second. Aged dried bone sample may becompletely milled after this protocol, but a few smaller fragments mayremain when using fresh bone. For fresh bone, the milled sample may becollected for further processing after the completion of milling cycle.Powderized bone or tooth samples may be transferred into sterile tubecontainers.

In other embodiments, the bone or tooth sample may be hammered andfragmented. Crude breakage of the bone or tooth may also be effective.For example, a piece of bone or tooth may be wrapped in a DurX®770(Berkshire Corporation, Great Barrington, Mass.) wipe (a non-woven sheetof polyester/cellulose material) or equivalent. Using a clean hammer ora similar hard, blunt object, e.g. a rock (washed and wiped down with anethanol wipe if available), the bone or tooth may be stroked severaltimes until the sample piece is broken into smaller pieces. Hitting thebone or tooth sample at different angles with the hammer may facilitatethe breakdown of the sample into smaller fragments. When hammering bone,small marrow pieces may be collected immediately. The harder bone bits(those refractory to initial hammering) can be hammered again intosmaller pieces by wrapping the bone pieces with a new DurX®770 wipe andstriking a few more times. The ideal size of bone fragments forprocessing is preferably about ⅛ inch or less. In general, the effect ofmilling or hammering is to generate small fragments and increase theexposed surface area of the bone accessible to further processing(thereby enhancing subsequent extraction and purification steps).

The ground or hammered bone or tooth samples may be mixed vigorously ina demineralization buffer to demineralize the bone matrix andconsequently, release DNA. In some embodiments, a demineralizationbuffer can be simply an EDTA solution with an EDTA concentration of 0.1Mto 1M, preferably 0.3-0.7M, more preferably 0.5M, without any otherchemicals. In other embodiments, the demineralization buffer, e.g., 0.5MEDTA, may further comprise a lysis buffer, DTT (0.02M) and proteinase K.In still other embodiments, the demineralization buffer, e.g., 0.5MEDTA, may further comprise a detergent, e.g., 0.5% SDS or 1% laurylsarcosinate or equivalent, or proteinase K (3-4 mg), or both.

The volume of a demineralization buffer added to samples may varydepending on the amount of the sample to be processed, its size, andtexture (hammered bone tends to absorb less than milled bone; aged driedbone tends to absorb more than fresher, red marrow-containing samples).FIG. 20 shows the average amount of purified DNA (in nanograms) as afunction of the volume of demineralization buffer added to a 500 mg bonepowder sample. The data was from quadruplicate measurements, with errorbars representing 1 standard deviation. This data demonstrates thatsufficient demineralization can be achieved with far less buffer thanused by prior art methods. For ≤10 mg milled or hammered bone, 120 μl ofbuffer is sufficient. For 200 mg bone, 200-220 μl of buffer issufficient. For 500 mg bone, 320-350 μl buffer is sufficient. Prior artmethods were based on the incorrect assumption that complete dissolutionof bone was required to allow DNA trapped in cells and bound tohydroxyapetite to be freed forensic samples and available for STRprocessing. We demonstrate herein that this is not the case and thatpartially demineralized bone using a simple demineralization solutionenables generation of excellent STR profiles. FIG. 21 shows ademineralization time-course. DNA yield/200 mg bone powder (innanograms) is plotted on the y-axis as a function of time on the x-axis.The data shows that overnight demineralization is not needed to obtainsufficient DNA for STR analysis. The data in the figure was from 6measurements, with error bars representing one standard deviation.

The invention may be designed to eliminate a long process of sampleconcentration without compromising pre-treatment efficiency and tomaximize the volume of liquid to be analyzed by conventional laboratoryor Rapid DNA analysis methodologies. In some embodiments, 300-350 μl ofa demineralization buffer, e.g., 0.5M EDTA, may be used for every 500 mgof bone or tooth; 150-220 μl of a demineralization buffer, e.g., 0.5MEDTA, may be used for every 100-200 mg of bone or tooth, and 100-150 μlof a demineralization buffer, e.g., 0.5M EDTA, for less than 100 mg boneor tooth.

This invention may be practiced with several demineralization buffers,including without limitation, 1% sarcosine, 0.002% SDS, 0.5% SDS andothers, with 0.5M EDTA being preferred. Prior art methods suggest that0.5% EDTA requires the use of detergents and proteinase K to obtainsufficient demineralization of bone to be useful in STR analysis. FIG.22 demonstrates that this is not the case. FIG. 22 demonstrates thatsimplified demineralization buffers of this invention result insufficient amounts of purified DNA for use in STR analysis.

The amount of the milled or hammered sample may be 1 mg-1 g, andpreferably 200-500 mg for aged/degraded samples with an I-Chip. Forfresh or intact bone samples in I-Chips, approximately 5-10 mg samplesmay be sufficient to obtain nucleic acids resulting in full STR profilesand less than 5 mg may generate full or useful partial STR profiles. Theactual DNA content of the sample affects the result. Nevertheless, adegraded bone sample with no intact DNA will not generate a profileregardless of how much sample is utilized. A full STR profile or auseful partial profile of a person means that the profile is sufficientto identify the person's identity and match when submitted to a databaseor utilized in a kinship algorithm. We report herein STR profilesgenerated using 16-locus and 27-locus assays, the latter showingsuperior sensitivity. With standard analytical scales, it is impossibleto measure minute samples, so one can adjust buffer volume to load ontoswab for analysis without signal saturation. The sample input in the27-locus STR profiles is theoretically equivalent to 0.6 mg sample.

Depending on the amount of genomic material expected to be present inthe sample (high or low), one may select the type of rapid DNA chip thatis best suited for processing. In the ANDE system, A-Chips are utilizedif it is estimated that 100's of ngs or more are present in a samplewhereas I-Chips are utilized if it is estimated that less than 100's ofngs of DNA are present. It follows that for hammered bone and teeth, forexample, I-Chips would typically be utilized for processing in the ANDERapid DNA system. The identical selection process can be utilized inother rapid DNA systems. In the event that too much or too little sampleis utilized (which can be determined by reviewing signal strength—peakheights—on the resulting electropherogram, repeat rapid DNA runsutilizing more or less starting material can be run. In addition, anAdaptive Expert System can be utilized to expand the dynamic range ofthe rapid DNA Identification system.

The quantity of DNA present in bone may be affected by several factorsincluding time of collection (fresh versus decomposed remains), with andwithout red marrow, the type of bone from the body, and the age andhealth of donor. Nevertheless, the selection of chip type may bestraightforward: relatively fresh bone may likely be processedeffectively in the high DNA content chip, and older bone may likely beprocessed effectively in the high DNA content chip.

The simple and rapid demineralization may be facilitated by vortexingthe mixture, for example, for 30 seconds to 1 minute on a benchtopvortex mixer (Vortex-Genie 2, Scientific Industries Model #G560) set atmax setting (scale 10). This demineralization step in the presence ofEDTA increases nucleic acid purification efficiency. In someembodiments, e.g., outside the laboratory, this vortexing may bereplaced with vigorous shaking by hand for at least 1 min and tappingthe tube several times to homogenize the contents.

In some embodiments, the liquid sample or the demineralized samplesolution may be allowed to settle until a supernatant forms under theinfluence of gravity. In other embodiments, the settlement may befacilitated by spinning the tube by hand. In still other embodiments,the liquid sample or the demineralized sample solution may be obtainedfrom the mixture, for example, by centrifugation. The centrifugation maybe for 2 minutes at 20,000×g. The supernatant (in the case of milledsamples) or sample liquid (in the case of hammered samples) contains theextracted nucleic acids.

In some embodiments, the supernatant containing the extracted nucleicacids may be used directly for further analysis without purification ina rapid DNA analysis system. In other embodiments, nucleic acids in thesupernatant may be further purified before being used for furtheranalysis.

Purification of nucleic acids from supernatant may be accomplished byvarious methods known in the art, for example, organic extraction usingphenol/chloroform/isoamyl alcohol (25:24:1), or silica purificationusing filters such as those from MagAttract® (Quiagen, N.V., Venlo, NL)DNA mini M48 kit.

In one embodiment, a small portion of the supernatant may be directlytransferred onto a transfer medium (e.g., a swab, pipette or filterpaper or the like), which may then be inserted into a rapid DNA analysissystem for nucleic acid analysis. The portion of the supernatant may beabout 100-150 μl. This invention may be practiced with a rapid DNAanalysis system capable of processing small portions or the supernatant,for example the ANDE™ system available from ANDE, Corporation, Waltham,Mass.). See e.g., Tan et al., Investig Genet. 2013; 4: 16 and Turinganet al., Investig Genet. 2016; 7: 2 and Grover et al., Int J Legal Med(2017) 131:1489-1501 which are herein incorporated by reference inentirety.

Kit for Nucleic Acid Extraction

In another aspect, the invention is a kit for performing the simplifiedand fast process for extracting nucleic acids from a nucleicacid-containing material, e.g., bone or tooth.

In one embodiment, the kit for extracting nucleic acids from bone andtooth comprises a buffer for demineralizing bone matrix and optionally abrochure detailing a rapid nucleic acid extraction process. In anotherembodiment, the demineralization buffer may contain only 0.5M EDTA. Insome embodiments, 0.5M EDTA may be combined with lysis buffer DTT(0.02M), and proteinase K. In other embodiments, 0.5M EDTA may becombined with detergent (0.5% SDS or 1% lauryl sarcosinate orequivalent) and proteinase K (3-4 mg).

In some embodiments, the brochure may include an instruction of how toperform nucleic acid extraction using the lysis buffer from bone andtooth samples. In some instances, the brochure may further describe howto pre-treat the bone and tooth samples. In still some instances, thebrochure may further describe how to analyze the extracted nucleicacids, for example, using a swab for analyzing the extracted nucleicacids in HDC BCS and/or LDC BCS system. In still some further instances,the brochure may describe the process for nucleic acid extraction suprain the present application.

In other embodiments, the kit may further include solutions for washingand cleaning the nucleic acid-containing material. In still otherembodiments, the kit may further include a swab for analyzing theextracted nucleic acids in HDC and/or LDC BCS.

Processes for Determining STR Profiles of Nucleic Acids from a Bone orTooth Sample

In yet another aspect, the invention is a process for determining STRprofiles of nucleic acids from a bone or tooth sample. In someembodiments, the process comprises providing the nucleic acid-containingmaterial in a form suitable for nucleic acid extraction; adding EDTA todemineralize the nucleic acid-containing material to obtain a mixture;mixing the mixture vigorously; separating the mixture to obtain a liquidsample that contains the extracted nucleic acids; subjecting a portion,if not all, of the liquid supernatant for fully integrated nucleic acidpurification and generation of an STR profile.

In some embodiments, bone and tooth samples may be fresh samples fromdonors with post-mortem interval (PMI) less than 8 hours or relativelyfresh samples from donors with PMI of 2-6 days. In other embodiments,bone and tooth may be from casework, disaster, missing persons orrelated samples. Such samples may be obtained as degraded as is thecase, for example, decayed bodies in fresh water, salt water, or soil orafter a fire or explosion.

In some embodiments, the samples are first cleaned and then grinded sothat the samples are in a form suitable for nucleic acid extraction. Forexample, the samples may be washed with water and scrubbed to removedebris, soaked in 10% bleach solution, and washed again with sterilewater before a final wash with 70% ethanol. After the final wash withethanol, the samples may be dried at room temperature and grinded, forexample, by freeze-mill to make powderized samples or hammering to makefragmented or granulated samples. Although not absolutely required,cleaning is advantageous in that it may remove large quantities ofmicrobial contaminants and, just as importantly, human DNA introduced byhuman handling or mixtures from two or more sample donors. Milling maybe also advantageous, but not necessary, consideration when the work isto be performed outside the laboratory.

In some embodiments, the bone or tooth sample may be milled andpowderized. By way of example, a piece of bone may be milled using manytechniques, for example using the SPEX Sample Prep 6870 LargeFreezer/Mill® following a 3-cycle program, 10 minute pre-cool, 2 minuterun time, 2 minute cool time, and with a rate of 10 cycles per second.Aged dried bone sample may be completely milled after this protocol, buta few smaller fragments may remain when using fresh bone. For freshbone, the available milled sample may be collected for furtherprocessing after the completion of milling cycle. Powderized bone ortooth samples may be transferred into sterile tube containers.

In other embodiments, the bone or tooth sample may be hammered andfragmented. Crude breakage of the bone or tooth may also be effective.For example, a piece of bone or tooth may be wrapped in a DurX®770 wipe(a non-woven sheet of polyester/cellulose material) or equivalent. Usinga clean hammer or a similar hard, blunt object, e.g. a rock (washed andwiped down with an ethanol wipe if available), the bone or tooth may bestroked several times until the sample piece is broken into smallerpieces. Hitting the bone or tooth sample at different angles with thehammer may facilitate the breakdown of the sample into smallerfragments. When hammering bone, small marrow pieces may be collectedimmediately. The harder bone bits (those refractory to initialhammering) can be hammered again into smaller pieces by wrapping thebone pieces with a new DurX®770 wipe and striking a few more times. Theideal size of bone fragments for processing is preferably about ⅛ inchor less. In general, the effect of milling or hammering is to generatesmall fragments and increase the exposed surface area of the boneaccessible to further processing (thereby enhancing subsequentextraction and purification steps).

The ground or hammered bone samples must be mixed vigorously in 0.5MEDTA to demineralize the bone matrix. In some embodiment, 0.5M EDTA maybe combined with lysis buffer and DTT (0.02M), and proteinase K (10 mg).In other embodiments, 0.5M EDTA may be combined with detergent (0.5% SDSor 1% lauryl sarcosinate or equivalent) and proteinase K (3-4 mg).

The volume of demineralization buffer added to samples may varydepending on the amount of the sample to be processed, its size, andtexture (hammered bone tends to absorb less than milled bone; aged driedbone tends to absorb more than fresher, red marrow-containing samples).The invention may be designed to eliminate a long process of sampleconcentration without compromising pre-treatment efficiency and tomaximize the volume of liquid to be loaded onto the swab for analysiswithout over saturation. In some embodiments, 300-350 μl of 0.5M EDTAmay be used for every 500 mg of bone or tooth; 150-220 μl of 0.5M EDTAmay be used for every 100-200 mg of bone or tooth, and 100-150 W of 0.5MEDTA for less than 100 mg bone or tooth. Tooth samples may requirelonger incubation than bone. In most cases, full STR profiles may begenerated from bone with 1 min incubation while full STR profiles fromtooth with 10 min incubation.

The amount of the milled or hammered sample can be 1 mg-1 g or more, andpreferably 200-500 mg for aged/degraded samples in I-Chip. Forfresher/intact bone samples in I-Chip, approximately 5-10 mg samples maybe sufficient to obtain nucleic acids resulting in full STR profiles andless than 5 mg may generate full or useful partial profiles. The actualDNA content of the sample will determine the result. Nevertheless, adegraded bone sample with no intact DNA will not generate a profileregardless of how much sample is utilized. A full STR profile or auseful partial profile of a person means that the profile is sufficientto identify the person's identity and match when submitted to a databaseor utilized in a kinship algorithm.

Depending on the amount of genomic material expected to be present inthe sample (high or low), one may select the type of chip, A-Chip orI-Chip, that is better suited for processing. The quantity of DNApresent in bone may be affected by several factors including time ofcollection (fresh versus decomposed remains), with and without redmarrow, the type of bone from the body, and the age and health of donor.Nevertheless, the selection of chip type may be straightforward:relatively fresh bone may likely be processed effectively in the highDNA content chip/A-Chip, and older bone may likely be processedeffectively in the low DNA content chip/I-Chip.

The simple and rapid demineralization may be facilitated by vortexingthe mixture, for example, for 30 seconds to 1 minute on a benchtopvortexer set at max setting (scale 10). This demineralization step inthe presence of EDTA may help increase nucleic acid yield becausedecrease in nucleic acid yield is observed without treatment with EDTA.In some embodiments, e.g., outside the laboratory, this vortexing may bereplaced with vigorous shaking by hand for at least 1 min and tappingthe tube several times to homogenize the contents.

In some embodiments, the liquid sample or the demineralized samplesolution may be allowed to settle until a supernatant forms under thelaws of gravity. In other embodiments, the settlement may be facilitatedby spinning the tube by hand. In still other embodiments, the liquidsample or the demineralized sample solution may be obtained from themixture, for example, by centrifugation. The centrifugation may be for 2minutes at 20,000×g. The supernatant (in the case of milled samples) orsample liquid (in the case of hammered samples) contains the extractednucleic acids.

In some embodiments, the supernatant containing the extracted nucleicacids may be used directly for further analysis without purification. Inother embodiments, nucleic acids in the supernatant may be furtherpurified before being used for further analysis.

Purification of nucleic acids from supernatant may be accomplished byvarious methods known in the art, for example, organic extraction usingphenol/chloroform/isoamyl alcohol (25:24:1), or silica purificationusing filters such as those from Qiagen MagAttract® DNA mini M48 kit.

Kit for Determining STR Profiles of Nucleic Acids from a Bone or ToothSample

In yet another aspect, the invention is a kit for determining STRprofiles of nucleic acids from a bone or tooth sample. In someembodiments, the kit may include a 0.5M EDTA as demineralization buffer;a brochure detailing a rapid nucleic acid extraction process; and a swabfor analyzing the extracted nucleic acids in LDC BCS.

In some embodiments, the kit may further include solutions for washingand cleaning the bone or tooth sample to remove adhered tissue debris onsurface, dirt, and exogenous DNA using water, followed by 10% bleachsolution, then rinsing again with water, and finally with 70% ethanol.

In some embodiments, the kit is for profiling the STR multiplexes whichinclude the following loci: D3S1358, TH01, D21S11, D18S51, Penta E,D5S818, D13S17, D7S820, D16D539, CSF1PO, Penta D, Amelogenin, vWA,D8S1179, TPDX, and FGA (the Powerplex 16 chemistry). In otherembodiments, the kit is for profiling STR multiplexes including thefollowing loci: D3S1358, D19S433, D2S1338, D22S1045, Penta B, TH01,D18S51, D1S1656, D10S1248, D2S441, Penta C, D16S539, vWA, D21S11,D12S391, Amelogenin, Penta D, D5S818, D13S317, D7S820, TPDX, CSF1PO,Penta E, D8S1179, FGA, SE33, DYS391, D6S1043, DYS439, DYS389II, DYS19,DYS392, DYS393, DYS389I, DYS390, DYS385a, DYS385b, DYS437, and DYS438.In still other embodiments, STR multiplexes will include the followingloci: 23 autosomal loci (D1S1656, D2S1338, D2S441, D3S1358, D5S81,D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51,D19S433, D21S11, D22S1045, FGA, CSF1PO, Penta E, TH01, vWA, TPDX, SE33),three Y-chromosomal loci (DYS391, DYS576, and DYS570), and Amelogenin.For all sample types discussed herein, there is no limit to the sets ofSTR multiplexes utilized for the generation of DNA IDs. Typically, atleast 7 STR loci should be used in the generation of DNA IDs because theRandom Match Probability with this number is sufficiently low (one intens of thousands to one in millions) to allow positive DNAidentifications. The specific STR multiplexed utilized is based on theapplication (e.g. search of a very large DNA database [ten million ormore samples as in the US, UK, and Chinese databases] to match a DNAfrom a potential rapist]; or search of a relatively small database[hundreds to thousands of samples for disaster victim identification ina plane crash]).

Processes for Simplified and Fast Semen DNA Analysis

In another aspect, the invention is a process for analyzing nucleicacids from a semen sample. A semen sample may be neat semen or a semenstain on a fabric. Alternatively, a semen sample may be directly orindirectly collected from an individual. In some instances, the semensample may be a forensic semen sample, e.g., a casework semen sample.Even though the form of a semen sample may vary, the present inventionis equally applicable as long as the semen sample may be collected ontoa swab head.

In some embodiments, the process for determining STR profiles of nucleicacids from a semen sample comprises the steps of collecting the semensample onto a swab, applying an appropriate amount of a dithiothreitol(DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solutiononto the swab head; and inserting the swab into a rapid nucleic acidanalysis system for STR analysis. The inventive processes herein may beused with any panel of STR loci. In some embodiments, the kit is forprofiling the STR multiplexes which include the following loci: D3S1358,TH01, D21S11, D18S51, Penta E, D5S818, D13S17, D7S820, D16D539, CSF1PO,Penta D, Am, vWA, D8S1179, TPDX, and FGA. In other embodiments, the kitis for profiling STR multiplexes including the following loci: D3S1358,D19S433, D2S1338, D22S1045, Penta B, TH01, D18S51, D1S1656, D10S1248,D2S441, Penta C, D16S539, vWFA31, D21S11, D12S391, Amelogenin, Penta D,D5S818, D13S317, D7S820, TPDX, CSF1PO, Penta E, D8S1179, FGA, SE33,DYS391, D6S1043, DYS439, DYS389II, DYS19, DYS392, DYS393, DYS389I,DYS390, DYS385a, DYS385b, DYS437, and DYS438. In still otherembodiments, STR multiplexes will include the following loci: 23autosomal loci (D1S1656, D2S1338, D2S441, D3S1358, D5S81, D6S1043,D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433,D21S11, D22S1045, FGA, CSF1PO, Penta E, TH01, vWA, TPDX, SE33), threeY-chromosomal loci (DYS391, DYS576, and DYS570), and Amelogenin.

In other embodiments, the semen sample may be neat semen or dried semensample on a piece of fabric and the process comprises the steps ofcollecting the nucleic acid-containing material sample onto a swab,applying DTT or TCEP to the swab, and subjecting the swab into the ANDEsystem, for example the I-Chip, for analysis. In some instances, thesemen sample may be dried semen stain on a fabric and the process mayfurther comprise the step of scraping the semen sample from the fabricwith a scraping tool, such as a knife, a razor blade or a scalpel. Insome instances, the semen stain on the fabric may be wetted with sterilewater and the swab rubbed and pressed against the fabric.

In some embodiments, the collection step may be carried out by scrapingthe fabric with a tool such as a knife, a razor blade or a scalpel. Thecollection step may be to collect the fabric fibers that the semensample has been dried on. In a preferred embodiment, the semen samplesare collected onto swabs. The swabs may be wetted with water beforebeing used to collect semen samples. In some instances, the swab may bewetted with a few drops of molecular grade water from a water dropper,preferably 1-2 drops. In other instance, the swab may be wetted withmore water due to the increased size of the swab head. In otherinstance, the stain on the fabric may be wetted with a few drops ofmolecular grade water from a water dropper, preferably 1-2 drops. Thewetting of the swab head and the fabric stain may facilitate thecollection of semen samples.

In some embodiments, the wetted swab may be used to swab the surface ofthe scraped fabric material. In other embodiments, the swab may befurther used to collect the clumps of the fabric fiber that waspreviously scraped off the fabric material. It is preferred to securethe clumps of fabric fibers onto the swab head before further processingthe swab.

In some embodiments, the swab head may be applied with 50 μl 150 mM DTTor 50 μl 50 mM TCEP. The presence of DTT or TCEP may facilitate thebreakdown of disulfide bonds on sperm nuclear membranes in the semensample. The application of the 150 mM DTT or 50 mM TCEP is preferablyapplied in a manner so that DTT/TCEP cover the entire swab head tofacilitate the recovery of nucleic acids.

In some embodiments, nucleic acids from the swabs may be extracted andanalyzed by using the I-Chip in the ANDE Rapid DNA Analysis System. TheI-Chip was developed by maximizing the efficiency of the purificationmodule and incorporating a post purification DNA concentration module.These modifications allowed the system to generate STR profiles fromsamples containing a few nanograms of DNA or less.

Kit for Determining STR Profiles of Nucleic Acids from a Semen Sample

In still yet another aspect, the invention is a kit for determining STRprofiles of nucleic acids from a semen sample. In one embodiment, thekit includes a DTT or TCEP solution, a brochure detailing a rapidnucleic acid extraction process from a semen sample as disclosed in thepresent application, and a swab used for collecting and analyzing theextracted nucleic acids in I-Chip. In some instances, the DTT solutionhas a DTT concentration equal or close to 150 mM and TCEP solution has aTCEP concentration equal or close to 50 mM. When the semen sample is asemen stain on a fabric, the kit may further comprise a tool, forexample, a knife, a razor blade, or a scalpel, for collecting semensamples from a fabric. The kit also contains a dropper bottle containingsterile water or an ampoule containing sterile water. In someembodiments, the kit may be used to generate profiles of the followingSTRs: D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13517, D7S820,D16D539, CSF1PO, PentaD, Am, vWA, D8S1179, TPDX, and FGA.

Further Analysis of Nucleic Acids Extracted Using this Invention

Extracted nucleic acids may be analyzed in various nucleic acid analysissystems, e.g., ANDE™ Rapid DNA Analysis System (ANDE Corporation). TheANDE™ System is a fully automated system with on-board expert systemsoftware for automated data analysis. The system can generate called STRprofiles from high DNA content samples (e.g. buccal swabs) inapproximately 90 minutes and from casework, sensitive site exploitation,and mass disaster samples (e.g. bloodstains, chewing gum, drinkingcontainers, clothing, touched items such as steering wheels, doorhandles, cell phones, and keyboards, liver, lung, brain, bone, andteeth) in approximately 105 minutes. The system is designed for use withthe high and low DNA content chips (“A-Chip” and “I-Chip” as referred toherein). The A-Chip and I-Chip consumables are “all-in-one” disposablecassettes, factory pre-loaded with all reagents needed for STR analysis.The A-Chip is designed for samples with large quantities of DNA—forexample, buccal swabs, and the I-Chip is designed for samples thattypically contain smaller quantities of DNA—for example, a small bloodstain, a cigarette butt, or a steering wheel swab. Both of theseconsumables are suitable for the processing of bone, teeth, semen, andother samples prepared using the teachings herein. The quantity of DNApresent in bone may be affected by several factors including PMI tocollection, condition of remains (e.g. decomposed remains, burned),presence or absence of marrow, the type of bone from the body, and theage and health of donor. Relatively fresh and intact bone may likelycontain greater quantities of DNA than older that has been exposed tothe environment or insult.

In one embodiment, a small portion of the supernatant may be directlytransferred onto a swab, which may then be inserted into a rapid DNAanalysis system, for example, either an A-Chip or I-Chip, for nucleicacid analysis. The portion of the supernatant may be about 100-150 μl.The swab may be an ANDE® swab or a similar swab which would fit the swabcap and swab chamber in a Rapid DNA Analysis system. The A-Chip isdescribed in Tan et al, Inv. Genetics 4 2013 and in Grover et al, Intg JLegal Med 2017; and the I-Chip in Turingan et al, Inv Genetics 7 2016,both of which are incorporated in their entirety herein.

Example 1. Extraction, Purification, and Analysis of Nucleic Acids fromBone

In this example, we performed a rapid nucleic acid extraction from bonesamples and analyzed the extracted nucleic acid using a swab and theI-Chip in the ANDE system.

Bone samples were artificially degraded to mimic casework and/ordisaster victim identification in plane crash tragedies (e.g. IndonesiaAirAsia Flight 8501). Mock decomposition of bone samples (femoral andhumeral fragments) was performed by soaking the fragments in Atlanticcoast salt water at 84° F. (average temperature of Java Sea) for 4 days.The bone samples were then washed with water, bleached, washed againwith water, and then ethanol. After drying, the bones were grinded byeither freezer mill to obtain powdered samples or simply hammered toobtain fragmented or granulated samples. The grinded bone samples wereplaced into four separate 2 ml centrifuge tubes containing 200 mghammered bone, 500 mg hammered bone, 200 g freeze-milled bone, and 500mg freeze-milled bone, respectively.

We then added 200-350 μl of 0.5M EDTA into each of the tubes. EDTA wasadded to demineralize the bone sample and help release DNA. The tubeswere then vortexed for 1 min using a benchtop vortexer at “10” speedsetting. The tubes were then centrifuged (Centrifuge 5417R; EppendorfNorth America, Hauppauge, N.Y., USA) at 20,000 rcf for 2 minutes toseparate the bone particulates from the demineralized supernatant. About150 μl of each supernatant was pipetted onto an ANDE swab for sampleinsertion into I-Chip for analysis of STR profiles. The STRs wereD3S1358, TH01, D21511, D18551, Penta E, D5S818, D13517, D7S820, D16D539,CSF1PO, Penta D, Am, vWA, D8S1179, TPDX, and FGA. As a result, we wereable to obtain STR profiles for each of the four test samples. See FIGS.1-4, nucleic acids extracted from each of the four test samples. FullSTR profiles (PP16) were generated from either milled or hammeredsamples and with decreased in bone input from 500 mg to 200 mg.

As a positive control, we performed nucleic acid extraction from 500 mgfreeze-milled bone from the same source following the totaldemineralization method reported in Loreille et al., Forensic ScienceInternational: Genetics 1 (2007) 191-195. Specifically, the bone powderwas incubated overnight in 7.5 ml of extraction buffer (EDTA 0.5M and 1%laurylsarcosinate) and 200 μl 20 mg/mL proteinase K in a rotary shakerat 56° C. Supernatant was transferred to an Amicon 30 centrifugal filterunit (Millipore) for concentration to approximately 150 μl and loadedonto a NetBio swab for a fully-integrated run in an I-CHIP. As a result,we were able to obtain a STR profile from the purified DNA. See FIG. 8.Full STR profile was generated using the long process of totaldemineralization. Overall signal strength was comparable to profilesgenerated from the simplified and fast bone analysis method.

In the traditional method, the demineralization buffer contains EDTA,detergent, and proteinase K, high initial buffer volume (7.5 ml for 500mg of powder) is used, the demineralization requires overnightincubation at 56° C. with agitation in a rotary shaker, and the processrequires sample concentration and washing using centrifugal units.

In contrast, in the present invention, the demineralization solution issimply an EDTA solution without any detergent or proteinase K. Inaddition, there is no requirement of incubation for extended period oftime or heating. Indeed, the demineralization process takes less than 5mins including sample handling and solution transfer. The resultantsupernatant contains the extracted nucleic acids that can be useddirectly to generate profiles using our HDC or LDC BCS and ANDE system.

Example 2. Extraction and Analysis of Nucleic Acids from Tooth

In this example, we performed a rapid nucleic acid extraction from toothsamples and analyzed the extracted nucleic acid in swab and LDC BCSsystem. The procedure is a modification of Example 1. After a 1 minutevortex, the tooth sample was incubated for 10 mins at 56° C. to enhancedemineralization. As a result, we were able to obtain STR profiles ofthe nucleic acids extracted from the tooth sample. See FIG. 9 showingSTR profile of the nucleic acids extracted from 200 mg freeze-milledtooth powder from root.

Our results indicate that a 1 minute demineralization process withtooth, was insufficient to obtain consistent full or good partialprofiles. This is probably due to the more compact matrix of toothcompared to bone and a longer demineralization is needed for DNArelease. The root of a tooth sample has been reported to contain moreDNA than the crown or other parts of the tooth. We notice that when weprocess crown, as opposed to root tissue from the same tooth sample, weare more consistently able to obtain full STR profiles from root and notfrom crown tissue. For fresh tooth or tooth subjected to degradation,root is preferred over crown. If only crown is available for processing,one may put in more material (say 500 mg instead of 200 mg) and milledis preferred over hammered.

FIG. 17 is a 16-locus full profile on 200 mg tooth root (undegraded);FIG. 18 is a 16-locus profile from tooth root aged 1 month 84 Fsaltwater (200 mg); FIG. 19 is the artificially aged molar which wascleaned in bleach and dried prior to cutting/milling.

Example 3. Extraction and Analysis of Nucleic Acids from Casework ToothSamples

Casework tooth samples were obtained from burnt bodies that had beenburied for at least 2 years. Tooth samples were washed in running waterand antibacterial soap while scraping the surface with cloth and bleach.The samples were then subjected to bleach-sterile-water-ethanol rinse,allowed to dry for 10 mins. The root was separated from the crown usinga dremel. The root was then freeze-milled and 200 mg was demineralized.

Example 4. Extraction and Analysis of Nucleic Acids from Neat Semen

Typically, semen samples contain ˜50 million cells per mL. After thawingon ice, the tube containing the semen sample was carefully opened andthe sample was gently mixed by pipetting up and down prior to sampleretrieval to ensure homogeneity of the sample. A 50 μl of semen diluted10-100 folds in 1×PBS buffer was pipetted on an ANDE swab. To the swab,a final concentration of 150 mM DL-Dithiothreitol (DTT) (Sigma Aldrich;Catalog #43816) or 50 μl of 50 mM Tris(2-carboxyethyl)phosphinehydrochloride (TCEP) (Sigma Aldrich; Catalog #646547) was added andincubated for 1-10 minutes at room temperature prior to analysis inI-Chip.

Example 5. Extraction and Analysis of Nucleic Acids from Dried Semen onFabric

In this example, we performed a rapid nucleic acid extraction from semensamples and analyzed the extracted nucleic acid in swab using theI-Chip. Here we used a mock semen sample which was prepared as describedin step 1 below. For casework semen samples, this semen samplepreparation step (step 1) is skipped.

Step 1: Preparing a Mock Semen Stained Clothing

Approximately 100 μl of neat semen was transferred onto a cotton fabricand/or denim. While the semen stain was still wet, the immediate areasurrounding the stain was marked with a permanent marker to ensure thatbiological materials were collected from the correct location, as thedried stain may not be visible to the unaided eye. The semen stain onfabric was let dry overnight at room temperature.

Step 2: Collecting Dried Semen Samples from Clothing

This step was carried out to collect DNA-containing semen sample fromthe semen stained fabric pieces. The piece of fabric was placed onto asterile workspace. We used a clean razor blade or sterile disposablescalpel to thoroughly scrape the entire marked stained fabric untilclumps of fabric fibers formed. An ANDE swab pre-wetted with 2-3 dropsof sterile water from a 30 mL drop-dispenser bottle (VWR; Catalog wasused was used to collect the DNA-containing semen samples. #16354-421).

As shown in FIG. 23, to collect DNA-containing semen samples, we movedthe clumps of fiber to one side of the stained fabric piece, and swabbedthe surface of previously scraped material by moving the swab head backand forth to collect residual cellular materials. Next, 1 drop ofmolecular biology grade water was placed onto the swab head andcollected the clumps of fiber. In some cases, an additional 1-2 drops ofmolecular biology grade water was placed onto the clumps of fiber toensure they are securely attached to the swab head. We then smoothed thesurface of the swab head with the razor blade or sterile scalpel tofurther secure the fibers. In addition, we also swabbed the razor bladeor sterile scalpel that was used to scrape the article of clothing ontothe dried portion of the swab head. Usually the dried portion of theswab head was closer to the plastic shaft. Finally, we loaded 50 μl of150 mM DL-Dithiothreitol (DTT) (Sigma Aldrich; Catalog #43816) or 50 μlof 50 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (SigmaAldrich; Catalog #646547) onto the fabric bundles and then rotating theswab to ensure coverage of the entire swab and return the swab with thefiber bundle to the clear plastic tube. The DTT or TCEP was used tobreak down the disulfide bonds in sperm nuclear membranes.

Step 3: DNA Analysis by Performing a Rapid DNA Run on the ANDE System

We processed and analyzed nucleic acids on the swab using the ANDEI-Chip. Specifically, we removed the protective plastic seal from thefirst swab chamber (found on the top of the I-Chip), selected “PerformRun” on the ANDE instrument, followed the screens for scanning the swabRFID using the swab caps only, entered the sample ID, and inserted theswab into the swab chamber by pressing down the swab until the swab capclicked securely into place.

As a result, full STR profiles of nucleic acids from the dried semen onclothing were successfully generated. Signal strength generated may varydepending on the donor and fabric material. See FIGS. 24 and 26 for thefull 16-locus and 27-locus STR profiles, respectively of nucleic acidspurified from dried semen samples on denim, and FIG. 25 for the full16-locus STR profile of nucleic acids purified from dried semen sampleson cotton.

Example 6. Extraction and Analysis of Nucleic Acids from TimedPost-Coital Vaginal Swabs and Swabs from Sexual Assault Kit (SAK)

Vaginal swabs from several donor pairs at 24 hr, 48 hr, and 72 hrpost-coital intervals were processed and analyzed. The SAK protocol issummarized in FIG. 27. The swab was placed in Tube 1 containing ANDE'sproteinase K and lysis solution. The sample was then vortexed for 10seconds and flashspun. The swab was manually transferred into a spinbasket that was inserted into Tube 1 and centrifuged for 5 minutes. Thiscentrifugation time can be reduced to 2 minutes. Centrifugation rids theswab of any residual liquid that may contain biological material. Thespin basket containing the dried swab was then removed and discarded.The aqueous phase is essentially the female fraction which containslysed epithelial cells. The female fraction was carefully pipetted outand transferred into another microfuge tube, Tube 2. For analysis of thefemale fraction in the I-Chip, 5-10 μl of the fraction was placed on anANDE swab. Sperm cells, if present, are pelleted at the bottom of theTube 1. It is therefore important not to disturb the pellet or remove itby accident. The pipette tip should not touch the bottom of the tube andliquid approximately 500 can be retained to avoid sperm cell sampleloss. The pellet with minimal liquid from the female fraction was thenwashed by adding a wash buffer containing MgCl₂ and CaCl₂ to the 1 mlline on Tube 1. The mixture was then vortexed for 5 seconds, centrifugedfor 2 minutes at 20,000×g to repelletize the sperm. Approximately 50 μlof aqueous phase was retained, the remainder was discarded. Homogenizedsperm was then transferred to Tube 3 containing 2000 U-10000 U ofnuclease; 2000 U is sufficient amount to degrade soluble DNA for thiswork with incubation for 10 minutes at 37 C. The reaction was quenchedby adding 20 μl of a Stop Solution containing 0.5M EDTA and incubationfor 10 minutes at 56 C; further reduction to 3 minutes at 56 C issufficient to deactivate the nuclease. The nuclease used in thisinvention is recombinant DNAseI (Sigma Aldrich; Catalog #04536282001).Finally, DL-Dithiothreitol (DTT) (Sigma Aldrich; Catalog #43816) orTris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma Aldrich;Catalog #646547) is added at a final concentration equal or close to 150mM DTT and 50 mM TCEP, respectively and vortexed for 5 seconds. Theresulting solution is the male fraction and can be collected with anANDE swab for analysis in I-Chip.

Note that the teachings herein are applicable to a wide range of othersamples. For example, the methods for vaginal swab processing can alsobe applied to separate male from female cells in cases or penile-oraland penile-anal penetration. Although the examples herein discussedsettings of male rapists and female victims, cases of male rapist-malevictim and female rapist-male victim are also seen, and these methodsare effective in such cases.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

What is claimed is:
 1. A process for extracting nucleic acids from abone or tooth sample, comprising of: a. providing the sample grindedinto powder or hammered into fragments; b. adding a demineralizationbuffer to the nucleic acid-containing material to obtain a mixturewherein the demineralization buffer consists essentially of a 0.3-0.7 MEDTA solution without any other chemicals; c. mixing the mixturevigorously; d. incubating the sample and the demineralization buffer forless than 60 minutes for a bone sample, and for less than 180 minutesfor a tooth sample; and e. separating the mixture to obtain a liquidsupernatant wherein the liquid supernatant contains the extractednucleic acids.
 2. The process of claim 1 wherein the separation stepdoes not involve concentrating the nucleic acid.
 3. The process of claim1, wherein the volume of demineralization buffer is 300-350 μl for every500 mg of bone or tooth, or 150-220 μl for every 100-200 mg of bone ortooth, or 100-150 μl for less than 100 mg of bone or tooth.
 4. Theprocess of claim 1 wherein the sample is a decomposed bone or toothsample.
 5. A process for extracting nucleic acids from a bone or toothsample, comprising: a. providing the bone or tooth sample grinded intopowder or hammered into fragments; b. adding demineralization bufferconsisting essentially of less than 500 μl of a 0.5 M EDTA solutionwithout any other chemicals to the nucleic acid-containing material toobtain a mixture; c. mixing the mixture vigorously; d. incubating themixture for at least one minute, but less than 10 minutes with orwithout subjecting the mixture to heat; and e. separating the mixture toobtain a liquid supernatant without concentration; wherein the liquidsupernatant contains the extracted nucleic acids.
 6. The process ofclaim 5, wherein the volume of demineralization buffer is 300-350 μl forevery 500 mg of bone or tooth, or 150-220 μl for every 100-200 mg ofbone or tooth, or 100-150 μl for less than 100 mg of bone or tooth. 7.The process of claim 5 wherein the sample has a weight of less than 10mg.
 8. The process of claim 5 wherein the sample is a decomposed bone ortooth sample.
 9. The process of claim 1 wherein the step of separatingthe mixture to obtain a liquid supernatant is achieved bycentrifugation.
 10. The process of claim 9 wherein the centrifugation isat a speed of 20,000 rcf and above for 2 minutes or longer.
 11. Theprocess of claim 1, further comprising a step of subjecting a portion ofthe liquid supernatant to a nucleic acid analysis.
 12. The process ofclaim 11 wherein the nucleic acid analysis comprises transferring theportion of the liquid supernatant to a DNA processing system.
 13. Theprocess of claim 1 wherein the step of mixing the mixture vigorously iscarried out by vortexing the mixture for about 30 seconds or longer. 14.A process for determining STR profiles of nucleic acids from a bone ortooth sample, comprising: a. providing the bone or tooth grinded intopowder or hammered into fragments; b. adding a demineralization bufferto the bone or tooth to obtain a mixture wherein the demineralizationbuffer consists essentially of a 0.3-0.7 M EDTA solution without anyother chemicals; c. mixing the mixture vigorously; d. incubating thesample and the demineralization buffer for less than 60 minutes for abone sample, and for less than 180 minutes for a tooth sample; and e.separating the mixture to obtain a liquid supernatant; wherein theliquid supernatant contains the extracted nucleic acids; and f.subjecting a portion of the liquid supernatant to a nucleic acidanalysis to determine the STR profile of the nucleic acids from the boneor tooth.
 15. The process of claim 14, wherein the volume ofdemineralization buffer is 300-350 μl for every 500 mg of bone or tooth,or 150-220 μl for every 100-200 mg of bone or tooth, or 100-150 μl forless than 100 mg of bone or tooth.
 16. The process of claim 14 whereinthe sample is a decomposed bone or tooth sample.
 17. The process ofclaim 14, wherein the step of separating the mixture to obtain a liquidsupernatant is achieved by centrifugation.
 18. The process of claim 17,wherein the centrifugation is at a speed of 20,000 rcf and above for 2minutes or longer.
 19. The process of claim 14 wherein the step ofmixing the mixture vigorously is carried out by vortexing the mixturefor about 30 seconds or longer.
 20. The process of claim 14, wherein thenucleic acid analysis comprises transferring the portion of the liquidsupernatant to a DNA processing system.
 21. The process of claim 20wherein at least seven STR loci are integrated.
 22. The process of claim14 wherein the demineralization buffer is 0.5M EDTA.